Amplifying local serotonin signaling prior to dry-off hastens mammary gland involution and redevelopment in dairy cows.

The monoamine serotonin (5-hydroxytryptamine, 5-HT) has been reported to inhibit milk protein gene expression and increase mammary epithelial cell (MEC) tight junction permeability after milk stasis. We hypothesized that increasing serotonin synthesis and signaling within the mammary epithelium before milk stasis would increase systemic and local involution markers, and downregulate the expression of milk protein and tight junction during involution, leading to more efficient tissue growth during the redevelopment phase. Herein, we examined the outcomes of increasing local mammary 5-HT synthesis before milk stasis on involution biomarkers, mammary gland microstructure, and gene and protein expression during the dry period. Multiparous Holstein cows were administered intramammary infusions (via the teat canal) of sterile water (CON, 4 mL/teat, n = 7) or 5-hydroxy-l-tryptophan (5-HTP, serotonin precursor, 20 mg/teat, n = 7) once daily for 5 d before dry-off (d 0). Blood, milk, and mammary secretions were collected and analyzed for components and metabolites. Mammary secretions were collected 12 h after the last milking and on d 1 to 4 during the dry period at 1200 h. Mammary gland biopsies were performed on d 4 (i.e., involution phase) and d 36 (i.e., redevelopment phase) of the dry period for histological and molecular evaluation. Milk protein and tight junction gene expression was quantified via real-time PCR. Hematoxylin and eosin staining, immunohistochemistry (Ki67), and immunofluorescence (serotonin, cleaved caspase 3) were performed to visualize tissue microstructure and to quantify serotonin intensity and cell turnover. Data were analyzed in SAS (SAS Institute Inc.) using 2-way ANOVA. After d 0, mammary secretions of 5-HTP cows had increased concentrations of 5-HT, lactoferrin, and bovine serum albumin. On d 1, 5-HTP cows had greater α-lactalbumin concentrations in plasma relative to CON. Serotonin intensity was increased in the mammary tissue of 5-HTP cows on d 4, relative to CON. On d 4, milk protein and tight junction gene expression was downregulated, MEC number was reduced, and cleaved caspase 3 protein was greater in mammary tissue of 5-HTP cows, relative to CON. On d 36, milk protein genes were upregulated, and the lumen:outer alveolar area and Ki67-positive cells were increased in the mammary tissue of 5-HTP cows, relative to CON. Amplifying serotonin signaling in the mammary epithelium before milk stasis at dry-off achieves greater apoptosis, leading to a reduction in MEC, allowing for greater cell proliferation, which results in more MEC during the redevelopment phase preceding the onset of lactation.

Methods for detecting heat stress in hutch-housed dairy calves in a continental climate.

Dairy calves exposed to solar radiation, elevated ambient temperature, and humidity are at risk of impaired welfare and productivity. Initial detection of thermal discomfort requires determination of optimal heat stress indicators and thresholds. Such values have recently been established in calves in chronic, subtropical, and acute continental environments but not in continuous, temperate conditions. Herein, the objectives were to determine associations between animal-based and environmental heat stress indicators and establish environmental breakpoints for hutch-raised dairy calves during a continental summer. From June to August, dairy calves (n = 63; 14 to 42 d of age) were individually hutch-housed and managed according to the dairy standard operating procedures in Arlington, Wisconsin. Calf respiration rates (RR), rectal temperatures (RT), shaved or unshaved skin temperatures (ST), and hutch internal and external air speed were measured thrice weekly at 0700 and 1400 h after a 15 min hutch restriction. Environmental indices including dry bulb temperature (Tdb), black globe temperature, and relative humidity were measured every 15 min, averaged hourly, and used to calculate temperature-humidity index (THI) using 8 different equations (THI1-8). Correlation and linear regression models were used to determine relationships within and between animal-based and environmental indicators. Environmental breakpoints were established using segmented regression models to estimate THI and Tdb thresholds for abrupt changes in animal responses. There were strong, positive correlations between animal-based indicators and Tdb or THI1-8, with the strongest association observed between unshaved ST and Tdb (r = 0.80). The linear regression of animal-based indicators with the best fit included Tdb or Tdb plus relative humidity and air speed. The threshold at which RR and RT began to rise was at a THI of 69 for both or at a Tdb of 21.0 or 21.5°C, respectively. No threshold was established for ST. Together, these outcomes indicate that Tdb is an appropriate measurement to detect thermal discomfort for calves in a temperate summer climate and individual hutch housing. Monitoring of calves is warranted before ambient temperature reaches 21.0°C, corresponding to RR of 40 breaths per minute and RT of 38.5°C, to promote calf comfort and reduce the risk of hyperthermia-related welfare and productivity consequences.

Late gestation hyperthermia: epigenetic programming of daughter's mammary development and function.

Exposure to stressors during early developmental windows, such as prenatally (i.e., in utero), can have life-long implications for an animal's health and productivity. The mammary gland starts developing in utero and, like other developing tissues and organs, may undergo fetal programming. Previous research has implicated factors, such as prenatal exposure to endocrine disruptors or alterations in maternal diet (e.g., maternal over or undernutrition), that can influence the developmental trajectory of the offspring mammary gland in postnatal life. However, the direct links between prenatal insults and future productive outcomes are less documented in livestock species. Research on in utero hyperthermia effects on early-life mammary development is scarce. This review will provide an overview of key developmental milestones taking place in the bovine mammary gland during the pre- and postnatal stages. We will showcase how intrauterine hyperthermia, experienced by the developing fetus during the last trimester of gestation, derails postnatal mammary gland development and impairs its synthetic capacity later in life. We will provide insights into the underlying histological, cellular, and molecular mechanisms taking place at key postnatal developmental life stages, including birth, weaning and the first lactation, that might explain permanent detriments in productivity long after the initial exposure to hyperthermia. Collectively, our studies indicate that prenatal hyperthermia jeopardizes the normal developmental trajectory of the mammary gland from fetal development to lactation. Further, in utero hyperthermia epigenetically programs the udder, and possibly other organs critical to lactation, yielding a less resilient and less productive cow for multiple lactations.

Effect of late-gestation heat stress in nulliparous heifers on postnatal growth, passive transfer of immunoglobulin G, and thermoregulation of their calves.

Youngstock such as nulliparous pregnant dairy heifers are not typically considered for active cooling, as they are thought to be more heat tolerant than mature cows. Recently, the benefits of heat stress abatement in pregnant heifers were studied, but the effect of in utero hyperthermia on the calf is still unknown. Herein, we aimed to investigate the effects of late-gestation heat stress in nulliparous heifers on the growth, immune, and thermoregulatory responses of their calves. Pregnant nulliparous dams were randomly selected for either active cooling (CL; fans and soakers; n = 15) or heat stress (HT; n = 16) 60 d before expected calving. After birth, respiration rate, rectal temperature, skin temperature, and sweating rate were obtained from their heifer calves at 24 h and on d 14, 28, 42, and 56. Blood samples and body weights were both collected for measurement of total protein and hematocrit and calculation of average daily gains, respectively. Gestation length was shorter in HT heifers (272 vs. 276 ± 1.28 d) relative to CL heifers. Birth weights, weaning weights, body weights up to d 56, and average daily gain from birth to weaning were not different between in utero HT (IUHT; n = 13) and in utero CL (IUCL; n = 12) calves. Apparent efficiency of absorption of immunoglobulin G tended to be lower in IUHT calves (26.3 vs. 42.7 ± 9.0%), and serum IgG concentrations from birth to d 56 were significantly lower in IUHT calves relative to IUCL calves (22.0 vs. 32.4 ± 4.47 g/L). Postnatal respiration rate, rectal temperature, sweating rate, total protein, and hematocrit were not different between in utero treatments. There was a tendency for IUHT calves to have lower skin temperature at 24 h (34.9 vs. 36.9 ± 1.05°C), and skin temperature tended to be elevated in IUHT calves at d 56 (29.6 vs. 27.9 ± 1.05°C). In summary, the lack of heat stress abatement during late gestation reduces gestation length of nulliparous heifers. Additionally, providing active cooling to nulliparous heifers during the prepartum period confers immune benefits to their offspring, although it does not translate to growth improvements.

Peripheral serotonin regulates glucose and insulin metabolism in Holstein dairy calves.

Peripheral serotonin regulates energy metabolism in several mammalian species, however, the potential contribution of serotonergic mechanisms as metabolic and endocrine regulators in growing dairy calves remain unexplored. Objectives were to characterize the role of serotonin in glucose and insulin metabolism in dairy calves with increased serotonin bioavailability. Milk replacer was supplemented with saline, 5-hydroxytryptophan (90 mg/d), or fluoxetine (40 mg/d) for 10-d (n = 8/treatment). Blood was collected daily during supplementation and on days 2, 7, and 14 during withdrawal. Calves were euthanized after 10-d supplementation or 14-d withdrawal periods to harvest liver and pancreas tissue. 5-hydroxytryptophan increased circulating insulin concentrations during the supplementation period, whereas both treatments increased circulating glucose concentration during the withdrawal period. The liver and pancreas of preweaned calves express serotonin factors (ie, TPH1, SERT, and cell surface receptors), indicating their ability to synthesize, uptake, and respond to serotonin. Supplementation of 5-hydroxytryptophan increased hepatic and pancreatic serotonin concentrations. After the withdrawal period, fluoxetine cleared from the pancreas but not liver tissue. Supplementation of 5-hydroxytryptophan upregulated hepatic mRNA expression of serotonin receptors (ie, 5-HTR1B, -1D, -2A, and -2B), and downregulated pancreatic 5-HTR1F mRNA and insulin-related proteins (ie, Akt and pAkt). Fluoxetine-supplemented calves had fewer pancreatic islets per microscopic field with reduced insulin intensity, whereas 5-hydroxytryptophan supplemented calves had increased islet number and area with greater insulin and serotonin and less glucagon intensities. After the 14-d withdrawal of 5-hydroxytryptophan, hepatic mRNA expression of glycolytic and gluconeogenic enzymes were simultaneously downregulated. Improving serotonin bioavailability could serve as a potent regulator of endocrine and metabolic processes in dairy calves.

Late-gestation heat stress abatement in dairy heifers promotes thermoregulation and improves productivity.

Multiparous, nonlactating pregnant cows are negatively affected by heat stress, but the effect of heat stress on more thermotolerant pregnant heifers has received less attention. Our objective was to characterize the effect of late-gestation heat abatement on thermoregulatory responses and subsequent milk production of nulliparous Holstein heifers. Pregnant heifers, blocked by body condition score (BCS) and predicted transmitting ability (PTA) for milk, were enrolled in either heat stress (HT, shade of freestall barn; n = 16) or cooling (CL, shade of freestall barn, water soakers, and fans; n = 15) environments during the last 60 d of pregnancy (~8 weeks). Rectal temperature (RT; thermometer), respiration rate (RR; breaths/min), sweating rate (SR; VapoMeter, Delfin Technologies, Kuopio, Finland), and skin temperature (ST; infrared thermometer) were measured thrice weekly from enrollment to calving. Vaginal temperature (VT; i-button intravaginal device) was measured every 10 min for 7 consecutive days at wk -8, -6, -4, and -2 relative to calving and averaged hourly. Daily thermoregulatory patterns assessed by SR and ST, were measured every 4 h over a 36-h time interval at wk -6, -4, and -2 relative to calving. Upon calving, milk, protein, and fat yields were recorded twice daily for 15 wk. The average temperature-humidity index (Hobo Pro temperature probe, Onset Computer Corporation, Pocasset, MA) in the barn during the precalving period was 77 (minimum of 72, maximum of 82). Only heifers that gave birth to a female calf (CL = 12, HT = 14) were included in the statistical analysis. In the precalving period, CL heifers had lower RR (44.3 vs. 60.0 ± 1.6 breaths/min), RT (38.7 vs. 38.8 ± 0.04°C), unshaved ST (34.7 vs. 35.3 ± 0.17°C), and unshaved SR (19.0 vs. 35.2 ± 1.9 g/m2h), relative to HT heifers. Additionally, VT was lower in CL heifers during wk -4, and -2, specifically during early morning and early afternoon hours. When measured over a 36-h time interval, ST and SR were lower in CL heifers, when compared with HT heifers for all weeks. Notably, ST was reduced overnight and SR was reduced during the daytime. Cooled heifers had higher milk yield (35.8 vs. 31.9 ± 1.4 kg/d), when compared with HT heifers. Similar to multiparous cows, our data indicate that actively cooling heifers in late pregnancy is effective in promoting thermoregulation and results in elevated milk production postcalving.

Methods for assessing heat stress in preweaned dairy calves exposed to chronic heat stress or continuous cooling.

Although dairy calves are more thermotolerant relative to mature cows, they are still susceptible to heat stress, as demonstrated by elevated physiological responses and reduced feed intake under high ambient temperature and relative humidity. However, indicators of heat stress have not been well-characterized in calves. Herein, we evaluated associations between environmental and thermoregulatory and productive animal-based indicators of heat stress in dairy calves exposed to chronic heat stress or continuous cooling in a subtropical climate. Holstein calves were exposed to heat stress (HT; shade of barn, n = 24) or continuous cooling (CL; shade of barn plus 2 fans, n = 24) from 2 to 42 d of age. Environmental indices, including ambient temperature, relative humidity, temperature-humidity index (THI), and wind speed, and animal-based indices, including respiration (RR), heart rate (HR), rectal (RT), and skin temperature (ST) were recorded thrice daily (0900, 1300, and 1900 h). Milk replacer (MI) and grain intakes were recorded daily from 15 to 42 d of age. Using segmented regression models, we then estimated THI thresholds for significant changes in physiological responses. We found a strong, positive correlation between animal-based indicators (except for HR, MI, and grain intakes) and ambient temperature and THI, with the highest correlation obtained with ST and THI (r ≥ 0.72). Ambient temperature and ST and ambient temperature or THI and MI were the only correlations that differed between treatments. The coefficient of determination (R2) obtained from regression analyses to model animal-based indicators was substantially improved by the inclusion of environmental indicators, with the greatest improvement achieved with THI. Overall, continuous cooling by fans promoted calf heat loss as CL calves had lower RR, RT, ST, and higher feed intake compared with HT calves. Temperature-humidity index breakpoints could be determined for RT (THI = 67), RR (THI = 65), and MI (THI = 82) in HT calves, and only for RR (THI = 69) in CL calves. Skin temperature variables had no detectable breakpoints in either treatment due to the strong linear relationship to THI. Collectively, our results suggest that ST is appropriate to estimate chronic heat stress and that THI is the best environmental indicator of heat stress in dairy calves raised in a shaded, subtropical environment. At a practical level, calves should be closely monitored when THI reaches 65 to 69 to minimize the risk of heat stress-related impairments.

Increasing serotonin bioavailability alters gene expression in peripheral leukocytes and lymphoid tissues of dairy calves.

Dairy calves are born with a naïve immune system, making the pre-weaning phase a critical window for immune development. In the U.S., 40-60% of dairy farms feed milk replacer to pre-weaned calves, which are devoid of bioactive factors with immunological roles. Serotonin is a bioactive factor with immunoregulatory properties naturally produced by the calf and present in milk. Human and rodent immune cells express the serotonin machinery, but little is known about the role of serotonin in the bovine immune system. Supplementing milk replacer with 5-hydroxytryptophan (serotonin precursor) or fluoxetine (reuptake inhibitor) increases serotonin bioavailability. We hypothesized that increased serotonin bioavailability promotes serotonergic signaling and modulates the expression of immune related genes in peripheral leukocytes and immune-related tissues of dairy calves. The present experiment targeted candidate genes involved in serotonin production, metabolism, transport, signaling and immune regulation. We established that bovine peripheral leukocytes express all known serotonin receptors, and can synthesize, uptake and degrade serotonin due to the expression of serotonin metabolism-related genes. Indeed, we showed that increasing serotonin bioavailability alters gene expression of serotonin receptors and immune-related genes. Further research will determine whether manipulation of the serotonin pathway could be a feasible approach to bolster dairy calves' immune system.

Late-gestation heat stress impairs daughter and granddaughter lifetime performance.

Records of late-gestation heat stress studies conducted over 10 consecutive years in Florida were pooled and analyzed to test the hypothesis that maternal hyperthermia during late gestation impairs performance of the offspring across multiple generations and lactations, ultimately impeding the profitability of the US dairy sector. Dry-pregnant multiparous dams were actively cooled (CL; shade of a freestall barn, fans and water soakers, n = 196) or not (HT; shade only, n = 198) during the last 46 d of gestation, concurrent with the entire dry period. After data mining, records of 156 daughters (F1) that were born either to CL (CLF1, n = 77) or HT dams (HTF1, n = 79) and 45 granddaughters (F2) that were born either to CLF1 (CLF2, n = 24) or HTF1 (HTF2, n = 21) were used in the analysis. Life events and daily milk yield for 3 lactations of daughters and granddaughters were obtained. Milk yield, reproductive performance, and productive life data were analyzed using MIXED and GLIMMIX procedures, and lifespan was analyzed using PHREG and LIFETEST procedures of SAS (SAS Institute Inc., Cary, NC). Milk production of HTF1 was reduced in their first (2.2 kg/d), second (2.3 kg/d), and third lactations (6.5 kg/d) compared with CLF1. More HTF1 were culled before first calving, and the productive life and lifespan of HTF1 were reduced relative to CLF1 (4.9 and 11.7 mo, respectively). The granddaughters (HTF2) born to HTF1 produced less milk in their first lactation (1.3 kg/d) relative to granddaughters (CLF2) born to CLF1. More HTF2 were culled before first breeding relative to CLF2; however, productive life and lifespan were not different between HTF2 and CLF2 animals. An economic analysis was then performed based on the number of heat stress days, dry cows per state, and the aforementioned impairments on daughters' lifespans and milk production. Collectively in the United States, the economic losses for additional heifer rearing cost, reduced productive life, and reduced milk yield of the F1 offspring were estimated at $134, $90, and $371 million per year, respectively. In summary, late-gestation heat stress exerts carryover effects on at least 2 generations. Providing heat abatement to dry-pregnant dams is important to rescue milk loss of the dam and to prevent losses in their progeny.

Carry over effects of late-gestational heat stress on dairy cattle progeny.

The impacts of late gestation heat stress on the dam and her subsequent lactation are well-recognized. However, more recent research has demonstrated the long-lasting and severe negative consequences on the in-utero heat-stressed progeny. Dairy calves born to late gestation heat-stressed dams weigh less at birth and up to one year of age and have compromised metabolism and immune function. In-utero programming of these offspring may coordinate alterations in thermoregulation, mammary development, and milk synthetic capacity at different developmental windows. Thus, prenatally heat-stressed dairy heifers will produce less milk across multiple lactations and have a lower herd survival rate, potentially negatively impacting the U.S. dairy economy. Dry period heat stress abatement strategies should be considered not only for the productivity and welfare of the pregnant dam but also for the developing calf.

Pre- and postnatal heat stress abatement affects dairy calf thermoregulation and performance.

Prenatal heat stress during late gestation exerts long-term effects on growth and productivity of the dairy calf. Further, direct exposure to heat stress during the preweaning period impairs calf thermoregulation and performance. We examined the effects of heat stress abatement during the prenatal period, postnatal period, or both on calf performance. We hypothesized that calves exposed to pre- and postnatal heat stress abatement would perform most optimally in terms of thermoregulation, growth, and health responses when compared with calves that are heat-stressed at any time in the pre- or postnatal periods. Holstein calves born to heat-stressed (HT) or cooled (CL) dams during late gestation (44 ± 5 d; prenatal HT or CL) were exposed to heat stress or cooling postnatally for 56 d (postnatal HT or CL), resulting in 4 treatments: HT-HT, HT-CL, CL-HT, and CL-CL; n = 12/treatment. Calves were administered 4 L of pooled colostrum and after 2 d of age allotted 10 L/d milk replacer and up to 3 kg/d concentrate in automatic feeder group pens (n = 6/pen). Postnatal cooling was achieved by 2 fans (average wind speed 2 m/s). Thermoregulatory responses (respiration rate and heart rate; rectal, body, and skin temperature), feed intake, growth parameters including average daily gain and medication events were recorded, and blood samples were collected weekly. Thermoregulatory responses were lower in postnatal CL calves compared with postnatal HT. In the afternoon, HT-HT calves had the highest respiration rate and rectal temperature, HT-CL calves had the lowest respiration rate, and CL-HT calves had the lowest heart rate compared with the other treatment groups. Prenatal CL calves weighed more at birth and weaning with a tendency for greater average daily gain compared with prenatal HT calves, whereas postnatal CL calves had increased milk replacer and concentrate intake and a tendency for reduced fever, infection, and total medication events relative to postnatal HT. Prenatal HT calves were esophageal tube fed more often than prenatal CL. Blood hematocrit and 24-h serum IgG concentration were greater in prenatal CL calves relative to prenatal HT. Prenatal heat stress abatement improves weight gain, hematocrit, and immunoglobulin transfer, whereas postnatal heat stress abatement modulates thermoregulatory responses, feed intake, and calf health. This study is the first to characterize the combined effects of pre- and postnatal heat stress or active cooling on the dairy calf.

Increasing serotonin bioavailability in preweaned dairy calves impacts hematology, growth, and behavior.

Peripheral serotonin has been shown to regulate important physiological functions such as energy homeostasis and immunity, particularly in rodent and humans, but its role is poorly understood in livestock species. Herein, we tested the safety and effectiveness of increasing serotonin bioavailability in preweaned dairy calves by oral supplementation of a serotonin precursor (5-hydroxytryptophan, 5-HTP) or a serotonin reuptake inhibitor (fluoxetine, FLX). Bull Holstein calves (21 ± 2 d old; N = 24) were fed milk replacer (8 L/d) supplemented with either saline as control (CON, 8 mL/d, n = 8), FLX (40 mg/d, approx. 0.8 mg/kg; n = 8), or 5-HTP (90 mg/d, approx. 1.8 mg/kg; n = 8) for 10 consecutive days in a complete randomized block design. Heart rate (HR), respiration rate, rectal temperature, and health scores were recorded daily. Hip height and body weight were measured at d 1, 5, and 10 relative to initiation of supplementation. Blood samples were collected once before the supplementation period (d 1), during the 10-d supplementation period (daily), and during a 14-d withdrawal period (d 2, 3, 4, 7, and 14 relative to initiation of withdrawal). Cerebrospinal fluid and muscle tissue were collected from a subset of calves (n = 12) that were euthanized after the 10-d supplementation or 14-d withdrawal period. Whole blood serotonin concentrations increased in 5-HTP calves and decreased in FLX calves compared with CON (P < 0.001), indicating that serotonin bioavailability was increased in both groups. Whole blood serotonin concentrations of 5-HTP and FLX calves returned to CON levels after 7 d of withdrawal. All calves grew and were considered healthy throughout the study. In fact, calves fed 5-HTP had higher average daily gain compared with CON (0.87 vs 0.66 ± 0.12 kg/d, P = 0.05). Calves fed FLX had lower HR (P = 0.02) and greater red blood cells and hemoglobin counts on d 10 of supplementation compared with CON (P < 0.01). After the 14-d withdrawal period, FLX was not detected in circulation of FLX calves, but was still present in the muscle tissue. Our results demonstrate that manipulation of the serotonin pathway by supplementing FLX or 5-HTP is a feasible and safe approach in preweaned dairy calves; however, it takes more than 14 d for FLX to be completely withdrawn from the body.